We have begun the development of a new class of chemical probes, membrane-impermeant, cleavable cross-linkers, which are designed to elicit information about the three-dimensional structure of membrane proteins in situ. The feature which distinguishes these reagents from other cleavable, protein cross-linkers is that they do not permeate biological membranes, so that one can experimentally pose not only the question of which subunit interacts with which others, but also the question of at which face of the membrane such interactions occur. This proposal is aimed at the further development of these probes and at their application to questions of human erythrocyte membrane protein structure. Reagent development will concentrate on the N-hydroxysulfosuccinimide active esters which we have recently introduced. We will rigorously determine the side chain specificity of such reagents, and determine their rates of hydrolysis and aminolysis under conditions appropriate to their use as protein modification reagents. We will also synthesize new reagents of this class required for our erythrocyte membrane protein studies. We have shown that we can cross-link subunits of the human erythrocyte anion exchange channel protein (band 3) to dimers at the extracytoplasmic membrane face. We will investigate the possible association at the extracytoplasmic membrane face of the anion channel with the cytoskeletal attachment site (band 2.1). We will also apply our techniques to questions of the oligomeric structure of non-coomassie blue staining glycoproteins at the extracytoplasmic face and to oligomeric proteins at the cytoplasmic face of the human erythrocyte membrane. The latter will include calmodulin-associated membrane proteins, in which calmodulin can be viewed as a transient subunit of an oligomer.